An amniotic fluid composition and method of using

ABSTRACT

A composition for treating a patient with a tissue disease or malformity has a composition containing amniotic fluid. The amniotic fluid has a quantity of gender specific amniotic fluid based on a gender of a fetal source. A method of treating a patient with a tissue disease or malformity comprises the steps of: identifying the tissue region to be treated and selecting a location to apply either topically or by injection or inhalation a composition containing amniotic fluid; selecting the composition containing amniotic fluid wherein the amniotic fluid has a quantity of gender specific amniotic fluid based on a gender of a fetal source allowing more specific targeted growth factors to be used for specific disease processes; and applying or injecting the composition at or into the selected location.

RELATED APPLICATIONS

The present invention is a division of U.S. Ser. No. 16/175,042 filed onOct. 30, 2018; which is a continuation in part of U.S. Ser. No.15/158,101 entitled “Methods And Composition For Treatment Of PenileDefects” filed on May 18, 2016, now U.S. Pat. No. 10,548,925 issued onFeb. 4, 2020; which is a division of U.S. Ser. No. 14/363,142 filed onJun. 5, 2014, now U.S. Pat. No. 10,751,374 issued on Aug. 25, 2020;which is a national stage entry of PCT/US2013/023830 filed on Jan. 30,2013 which claims priority to 61/592,108 filed on Jan. 30, 2012 and acontinuation in part of U.S. Ser. No. 15/541,988 entitled, “Methods AndCompositions For Treatment Of Penile Defects” filed on Jul. 6, 2017which is a national stage entry of PCT/US2016/016934 filed on Feb. 8,2016 which claims priority to 62/112,709 filed on Feb. 6, 2015.

TECHNICAL FIELD

The present invention relates to a composition and method of treatingtissue disease or malformities in the tissue or treating diseaseprocesses by having the composition include growth factors found inamniotic fluid, more particularly a gender differentiated amnioticfluid. The Invention relates to being able to treat different specificdiseases by different types of Amniotic Fluid that is rich in differentspecific growth factors. The invention relates to the selectivityprocess that allows us to harness more specific growth factors forspecific diseases and is unique and never done before.

BACKGROUND OF THE INVENTION

The use of amniotic fluid to treat a variety of tissue disorders is wellknown and commonly practiced. There are however, a number of indicationsand treatments where the use of amniotic fluid has not been attemptedfor a variety of reasons. There are a number of areas where the resultsof treatments have been at best unpredictable for a variety of reasonsbased mostly on speculation and conjecture. As a result, the use ofamniotic fluid has had mixed reviews.

Within the uterus of a pregnant woman, a growing fetus is surrounded andcushioned by amniotic fluid, a watery liquid within the amnion Amnioticfluid is one of the main samples used for the medical examination of thepregnant woman and her fetus.

Traditionally, during a Caesarean section, after cutting through theuterus, the amniotic fluid will be suctioned away and discarded to makemore room Amniotic fluid contains cells, electrolytes, growth factors,carbohydrates, lipids, proteins, amino acids, lactate, pyruvate, enzymesand hormones Amniotic fluid is also a source of stem cells which ideallyshould be isolated and separately cultivated for cell therapy purposes.While amniotic fluid cells can be obtained from a small amount of fluidduring amniocentesis, these amounts are insufficient for a larger scaleharvesting of biomolecules or culturing of the stem cells comprisingamniotic fluid.

In US 2015/0025366 entitled “Method for Obtaining Sterile Human AmnioticFluid and Uses Thereof” published Jan. 22, 2015 stated, there is arecognized need in the art for an improved means for obtaining sterileamniotic fluid for use in research and the development of therapeuticproducts. Particularly, the prior art is deficient in methods forobtaining sterile human amniotic fluid with minimal or no risk to apregnant woman or fetus by collecting the amniotic fluid prior to anelective Caesarean section. Also, the prior art is deficient in methodsfor obtaining sterile human amniotic fluid devoid of cells which maycreate unwanted reactions due to their allogenic characters on thepatients to be treated. The inventor, Harrell, claimed to fulfill thislongstanding need and desire in the art to improve the safety ofamniotic fluid in its medical uses and taught how to sterile filter thefluid.

This prior art method of obtaining sterile filtered human amniotic fluidfrom an individual comprises the steps of obtaining sterile humanamniotic fluid from an individual, removing cells, large particles andother undissolvables from said human amniotic fluid by high speedcentrifugation, followed by membrane filtration. The first step is tocentrifuge the amniotic fluid in swing out buckets adapted to swing outrotors or other centrifugation bottles in angle rotors at about 5,000rpm to about 10,000 rpm for about 30 minutes to about 60 minutes. Thesupernatant is then filtered using filters with a pore size of about 5μm to about 10 μm to obtain the first filtrate, then filtering saidfirst filtrate through filters with a pore size of about 1.0 μm toobtain a second filtrate, filtering the second filtrate through filterswith the pore size of 0.45 μm or/and 0.2 μm to obtain a sterilelyfiltered amniotic fluid. In case of a final membrane filtration limitedto 0.45 μm, it is preferable to repeat a second filtration on a second0.45 μm membrane, to increase the sterility assurance level. The sterileamniotic fluid retains the growth factors from the raw amniotic fluid.In this method, the first centrifugation step may be replaced by depthfiltration through available filtration systems, however this option isnot preferred because it leads to important volume losses andundesirable adsorption of growth factors by the filtration media.

This technique disclosed by Harrell achieved a sterile fluid thatremoved all particles down to 0.2 micron. All cells and particulategreater than the 0.2 micron are removed, leaving a fluid devoid of muchof the beneficial biochemical particles needed to be useful in medicaltreatments.

An improved method of recovering amniotic fluid is described in US2017/0042943 A1 entitled “Liquid Amnion Transplant Product”, thatinvention discloses a method to recover amniotic fluid and maintainparticles in sizes up to 170 microns to 260 microns, over 100 timesgreater, but maintaining a non-immunogenic product having superiorbiochemical properties suitable for direct injection into patients.

The present invention is believed to have established, at least in sometreatment protocols, a novel and heretofore unexpected way to maximizethe use of recovered amniotic fluids suitable for direct injection or asa topical ointment that will be more reliable and have better positiveoutcomes.

One area of interest is that of penile defects, such as erectiledysfunction (ED) and Peyronie's disease. At least some forms of ED tracetheir etiology to vascular deficits in the penile tissue. Pharmaceuticalcompositions for treating ED do exist; however, a need exists for moreadvanced treatments that may require use of medicaments. At least onegroup has reported success in the use of stem cells derived from bonemarrow to reverse ED in an animal model. Kendirci M et al.Transplantation of non-hematopoietic adult bone marrow stem/progenitorcells isolated by the p75 nerve growth factor receptor into the penisrescues ED in a rat model of cavernous nerve injury. J. Urol. 2010;184(4): 1560-1566. Another group has seen success in usingmuscle-derived stem cells in a rat model of ED. Woo J C et al.Transplantation of muscle-derived stem cells into the corpus cavernosumrestores erectile function in a rat model of cavernous nerve injury.Korean J. Urol. 2011; 52: 359-363.

Peyronie's disease is an ailment involving the growth of fibrous plaquesin the soft tissue of the penis. These plaques grow in the tunicaalbuginea, a region of connective tissue that is part of the moregeneral connective tissue of Buck's fascia. The tunica albuginea is anarea surrounding the corpora cavernosa. The disease results in pain, ED,and alteration of penis shape, and may be caused by disorganizedcollagen fibers. Treatments for the disease currently includeadministration of corticosteroids to the plaques, radiation therapy, andvitamin E. Pharmaceuticals for treatment exist; however, the use ofmedicaments is considered controversial in the art, and these compoundshave not met with sustained success. Hauck E W et al. A criticalanalysis of nonsurgical treatment of Peyronie's disease. EuropeanUrology 2006; 49(6): 987-97.

To take full advantage of these opportunities, improved capabilities areneeded.

In order to correct penile defects such as Peyronie's disease orerectile dysfunction, it is known to inject medications into the tissueof the patient's penis. This procedure can typically be done in adoctor's office under supervision of a trained professional. It isimportant when injecting the penis that the medications be delivered tothe locations in need of treatment while avoiding areas that could causedamage.

Many of the new medications used during these procedures can includebiological products such as stem cells or other organic material thatneeds to be strategically placed along the shaft of the penis.

In some cases, it may be possible for the patient to self-inject himselfwith medications to assist in the repair and recovery process. Theself-injections will most likely occur at the patient's home withoutsupervision of a trained professional. This is very similar to diabeticpatients who routinely inject themselves with insulin. In such cases, itis important that the medications be delivered in the proper dosage atthe proper location. In some cases, the medication can be topicallyapplied generally, however, implantation or injections seem to be mosteffective.

The present invention provides a unique method and composition thatutilizes amniotic fluid as a critical source to deliver tissueregenerative proteins and growth factors to the tissue in need ofrepair. The present invention defines a unique selection process tomaximize the certain growth factors for certain disease processes. Thepresent invention summarized and described hereinafter provides a uniqueselection process and use and refinement in treatments employingamniotic fluid.

SUMMARY OF THE INVENTION

A composition for treating a patient with a tissue disease or malformityhas a composition containing amniotic fluid. The amniotic fluid has aquantity of gender specific amniotic fluid based on a gender of a fetalsource. From this gender source we are now able to get specific growthfactors in higher concentrations.

The gender specific amniotic fluid of female fetal source and the malefetal source exhibit about 1451 growth factors and cytokines when testedunder mass spectrometry. The female fetal source amniotic fluid exhibits49 growth factors in a statistically significant higher concentrationthan the male fetal source amniotic fluid. The male fetal sourceamniotic fluid exhibits 45 growth factors in a higher concentration thanthe female fetal source amniotic fluid. Each of the number of growthfactors concentration higher in each gender specific amniotic fluid isgreater than 25 and less than 50 of the total of about 1500. The femalefetal source amniotic fluid has 49 growth factors statisticallysignificantly greater than the male fetal source amniotic fluid. Themale fetal source amniotic fluid has 45 growth factors statisticallysignificant greater than the female fetal source amniotic fluid. Theselection of the composition with amniotic fluid is guided bydifferences in growth factors and cytokines between the gender specificamniotic fluids. The gender specific amniotic fluid has a selectedproportion of male fetal source amniotic fluid or female fetal sourceamniotic fluid relative to the total amniotic fluid. The selectedproportion of male fetal source or female fetal source amniotic fluid isbetween 75 percent and up to 100 percent and at 100 percent the amnioticfluid is either all female fetal source or all male fetal sourceamniotic fluid. The amniotic fluid is further selected based on diseaseprocess based upon age of term pre planned C-Sections in addition togender of fetus.

A method of treating a patient with a tissue disease or malformitycomprises the steps of: identifying the tissue region to be treated andselecting a location to apply either topically or by injection acomposition containing amniotic fluid; selecting the compositioncontaining amniotic fluid wherein the amniotic fluid has a quantity ofgender specific amniotic fluid based on a gender of a fetal source; andapplying or injecting the composition at or into the selected location.

Definitions

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines or type of stem cell,constructs, additives, and reagents described herein. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

The term “amniotic fluid” refers to the protective liquid contained bythe amniotic sac of a gravid Amniote. This fluid serves as a cushion forthe growing fetus, but also serves to facilitate the exchange ofnutrients, water, and biochemical products between mother and fetus.Recent studies show that amniotic fluid contains a considerable quantityof stem cells. These amniotic stem cells are pluripotent and able todifferentiate into various tissues, which may be useful for future humanapplication. Some researchers have found that amniotic fluid is also aplentiful source of non-embryonic stem cells. These cells havedemonstrated the ability to differentiate into a number of differentcell-types, including brain, liver and bone.

The term “growth factor” refers to a naturally occurring substancecapable of stimulating cellular growth, proliferation, healing, andcellular differentiation. Usually it is a protein or a steroid hormone.Growth factors are important for regulating a variety of cellularprocesses.

The term “protein” refers to large biomolecules, or macromolecules,consisting of one or more long chains of amino acid residues. Proteinsperform a vast array of functions within organisms, including catalysingmetabolic reactions, DNA replication, responding to stimuli, andtransporting molecules from one location to another. Proteins differfrom one another primarily in their sequence of amino acids, which isdictated by the nucleotide sequence of their genes, and which usuallyresults in protein folding into a specific three-dimensional structurethat determines its activity.

Growth factors typically act as signalling molecules between cells.Examples are cytokines and hormones that bind to specific receptors onthe surface of their target cells.

The term “stem cell” refers to any multipotent or pluripotent cell,traditional stem cells, progenitor cells, preprogenitor cells, andreserve cells. The term is used interchangeably with and may meanprogenitor cell. The stem cell may be derived from an adult organism orfrom a cell line, or from an embryonic organism. Exemplary stem cellsinclude embryonic stem cells, adult stem cells, pluripotent stem cells,neural stem cells, liver stem cells, muscle stem cells, muscle precursorstem cells, endothelial progenitor cells, bone marrow stem cells,chondrogenic stem cells, lymphoid stem cells, mesenchymal stem cells,hematopoietic stem cells, and the like. Descriptions of stem cells,including method for isolating and culturing them, may be found in,among other places, Embryonic Stem Cells, Methods and Protocols, Turksened., Humana Press, 2002.

The term “adult” as used herein refers to any non-embryonic organism.For example the term “adult adipose-derived regenerative cell,” refersto an adipose-derived regenerative cell, other than that obtained froman embryo.

The term “embryo” as used herein refers to any multicellular diploideukaryote during development, until birth or hatching. The term“embryonic stem cell” refers to a pluripotent cell derived from theinner cell mass of a blastocyst.

The term “mesenchymal stem cell” refers to any multipotent stromal cellderived from, for example and without limitation, umbilical cord blood,adipose tissue, muscle, corneal stroma, and dental pulp that candifferentiate into cells such as, including but not limited to,osteoblasts, chondrocytes, and adipocytes.

The term “adipose-derived regenerative cell” (ADRC) is usedinterchangeably with adipose stem cells (ASC) herein and refers to adultcells that originate from adipose tissue. ADRC are a heterologouspopulation of cells comprising at least one of the following populationof cells; adult stem cells, vascular endothelial cells, vascular smoothmuscle cells, endothelial cells, mesenchymal stem cells, fibroblasts,pericytes and additional other cell types.

In some embodiments, ADRC refers to a substantially pure population ofadipose-derived stem cells. ADRC can be easily harvested from adiposetissue and are substantially free of adipocytes and red blood cells andclonal populations of connective tissue stem cells. The stromal vascularfraction cells are substantially devoid of extracellular matrix materialfrom adipose tissue. ADRC may also be referred to as adipose-derivedstem/stromal cells (ASCs), adipose-derived adult stem (ADAS) cells,adipose-derived adult stromal cells, adipose-derived stromal cells,adipose stromal cells, adipose mesenchymal cells, adipose-derivedmesenchymal stem cells, lipoblasts, pericytes, preadipocytes, andprocessed lipoaspirate cells.

The term “adipose” as used herein refers to any fat tissue from asubject. The terms “adipose” and “adipose tissue” are usedinterchangeably herein. The adipose tissue may be brown fat, white fator yellow fat or white adipose tissue, derived from subcutaneous,omental/visceral, mammary, gonadal, or other adipose tissue site. Theadipose tissue has adipocytes and stroma. Adipose tissue is foundthroughout the body of an animal. For example, in mammals, adiposetissue is present in the omentum, bone marrow, subcutaneous space, andsurrounding most organs. Such cells may comprise a primary cell cultureor an immortalized cell line. The adipose tissue may be from anyorganism having fat tissue.

Preferably, the adipose tissue is human; most preferably, the adiposetissue is derived from the individual in need of treatment for a peniledefect. A convenient source of adipose tissue is from liposuctionsurgery, however, the source of adipose tissue or the method ofisolation of adipose tissue is not critical to the invention, andacquisition of adipose tissue by any means may adequately provide tissueand stem cells for the present invention.

The term “tissue” as used herein is a broad term that is applied to anygroup of cells that perform specific functions, and includes in someinstances whole organs and/or part of organs. A tissue need not form alayer, and thus encompasses a wide range of tissue, including adiposetissue derived from any source in an organism. Preferably, the tissue isderived from a mammal. Most preferably, the tissue is derived from theindividual in need of treatment for a penile defect.

The term “implant” as used herein refers to any method for transferringa population of cells or cell mass into a subject, including by surgicalimplantation (incision into the tissue of interest and depositiontherein) and injection by a syringe, needle, cannula, or the like of anysuitable gauge. An implant as used herein can comprise geneticallymodified cells, as well as cells differentiated from other cells, suchas stem cells, progenitors, and the like, as well as adipose cells ortissue.

The term “corpus cavernosum” of the penis refers to one of a pair ofsponge-like regions of erectile tissue which contain most of the bloodin the penis during penile erection. Generally, the two corpuscavernosum and a corpus spongiosum are three expandable erectile tissuesalong the length of the penis which fill with blood during erection. Theterm “corpus” is used interchangeably herein with corporal, corporealand corporic, which are terms used to describe tissues which are derivedfrom the corpora cavernosum or which can be developed, differentiated,or altered by natural or artificial means into corpora cavernosumtissue. The term “cavernosum” is used interchangeably herein ascavernae, corporum, cavernosum, or cavernosorum penis, and refers to thecaverns of corpora cavernosa (or one of the two corpus cavernosum) ofthe penis or the dilatable spaces within the corpus cavernosum of thepenis, which fill with blood and become distended with erection.

The term “tunica albuginea” refers to the fibrous tissue covering, orenveloping, the corpora cavernosa of the penis. This tissue consists ofelastin and collagen. The term “Bucks fascia” refers to the layer offascia covering the penis, including the tunica albuginea.

The terms “subject”, “individual” and “patient” are used interchangeablyherein, and refer to an animal, for example a human, from whom a adiposetissue and stem cells, for example ADRC can be harvested, or a subjectinto whom tissue can be transplanted for treatment, for exampletreatment for penile defects, using the compositions and methodsdescribed herein. For treatment of conditions or disease states whichare specific for a specific animal such as a human subject, the term“subject” refers to that specific animal. In some embodiments, thesubject is a human subject. It is possible in embodiments of thisinvention that recipient subjects are of a different mammalian subjectthan the donor subject.

The term “exosomes” refers to cell-derived vesicles that are present inmany and perhaps all eukaryotic fluids, including blood, urine, andcultured medium of cell cultures. Evidence is accumulating that exosomeshave specialized functions and play a key role in processes such ascoagulation, intercellular signaling, and waste management.Consequently, there is a growing interest in the clinical applicationsof exosomes. Exosomes can potentially be used for prognosis, fortherapy, and as biomarkers for health and disease.

The term “M RNA” refers to a large family of RNA molecules that conveygenetic information from DNA to the ribosome, where they specify theamino acid sequence of the protein products of gene expression.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by way of example and with reference tothe accompanying drawings in which:

FIG. 1 is a table showing the PSV wherein Female amniotic fluid wasstatistically significantly better than the male in treating ErectileDysfunction and improving Penile Peak Systolic Velocity (P<0.05),P=0.012.

FIG. 2 is a perspective view of a syringe with the amniotic fluidsuitable for direct injection.

FIG. 3 is a volcano plot graph showing male_1 way versus female (s/n)Volcano.

FIG. 4 is a volcano plot graph showing male_1 way versus female (s/n)MvA.

FIG. 5 is a graph showing standard deviation log 2 (FC).

FIG. 6 SD male_1 way versus female (s/n) Volcano.

FIG. 7 is a PCA graph.

FIG. 8 is a PCA graph.

FIG. 9 is a PCA graph.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a unique use of recovered amnioticfluid for treatment of various tissue conditions. The current use ofamniotic fluid for soft tissue is known in the art as disclosed in thepublication US 2017/004943 A1 which is being incorporated by referencein its entirety in the present invention. The processing of amnioticfluid occurs in a variety of ways, many of the process steps can damagethe amniotic fluid and reduce its potential for maintaining itsbiological activity in particular its growth factors. The process ofremoving debris and unwanted particles from amniotic fluid as a resultcan also cause a loss of the biologic beneficial effects of thematerial. In some cases, the material is processed to a point that issimply represents virtually sterile water when completed and may have nomedical value. The present invention, on the other hand, is relying onthe use of amniotic fluid that has been processed in such a way that thematerial will maintain its biological growth factors that are commonlyfound in the native amniotic fluids. What is unique about the presentinvention is that this invention establishes that there are differencesin amniotic fluids based on the fetal source. In particular, amnioticfluid is recovered from a mother planning to have a live birthpre-planned C-section. The amniotic fluid is recovered typically at 38weeks from the pregnant mothers and provided in batches forsterilization. It is believed that this timing is somewhat arbitrary andcould vary within a few weeks one side or the other of 38 weeks, forexample 36 to 40 weeks could also provide amniotic fluid. In any event,the source of the amniotic fluid has always been simply the fluidsurrounding the fetus that is withdrawn and typically used to bediscarded and is now provided for use in soft tissue treatments andother treatments.

What was of interest to the present inventor was whether or not therewas any significant difference between the amniotic fluids of a malefetus vs a female fetus. In other words, there was a speculation by theinventor that it may make a difference whether or not the fetal sourceof the amniotic fluid was a male or female. As a result of this, theinventor investigated sampling amniotic fluid that was selected based onthe gender of the fetus. Although Amniotic Fluid has been around sincethe inception of humans, and Amniotic Fluid as a treatment has beenaround for close to 10 years, no one has ever looked at the fetal Genderas being a factor that differentiates the amniotic fluid. And itsability to treat diseases as a whole or certain diseases. This is notobvious and very unique. In doing so, it was discovered that recoveredamniotic fluid analysed using mass spectrometry had about 1500 growthfactors and cytokines discovered. To be exact 1451 were reported in thisstudy. In this study, it was also reported that there were 49 growthfactors that were statistically significantly more present in FemaleAmniotic Fluid and 45 growth factors statistically significantly morepresent in Male amniotic fluid. Accordingly, it was established thatthere is a difference in the amniotic fluid based on the gender sourceof the fetus. This is a significant finding in that the amniotic fluidfrom one gender source may achieve beneficial results in certaintreatments superior to those of the opposite gender. This has never beenconceived of or done before in the literature.

To test this theory, the inventor decided to treat patients' penises whoexhibited either erectile dysfunction or Peyronie's disease. In doingso, the amniotic fluid was infected using a blind study wherein theurologist and the physician injecting had no idea which gender fetalsource the amniotic fluid came from, but only provided direct injectionsinto the penis. The penile peak systolic velocities of the patients'pre-injection and post-injection were tested. In the study it wasdetermined that the amniotic fluid based on the gender of the fetalsource of the amniotic fluid provided different growth factors thatwould have an effect on erectile dysfunction and Peyronie's disease. Tothe surprise of the inventor, the gender based amniotic fluid did have avery unexpected result in which the inventor found that female amnioticfluid was statistically significantly better than the male amnioticfluid in treating Erectile Dysfunction and improving Penile PeakSystolic Velocity (P<0.05), P=0.012. To the inventor, who is skilled inthe art, this was incredible and unobvious and extremely novel.

Similarly, the inventor discovered that the female sourced amnioticfluid seemed to improve Peyronie's disease better. However, enoughpatients have not been tested to date to establish a clear confirmationthat this is true. However, it is believed to have a similar beneficialeffect superior to the effect of the male sourced amniotic fluid. InPeyronie's Disease, Matrix Metalloprotinease Inhibition is important butcan be stopped by TGF Beta. Female Amniotic Fluid has more tissueinhibitors of matrix metalloproteinase (TIMP) expression than male andmay play an important role in helping with Peyronie's Disease. Also MaleAmniotic Fluid works on the TGF Beta receptor so may play equally asimportant a role.

The inventor believes that the use of amniotic fluid differentiated bythe gender of the fetus has never been done before Amniotic fluid thatwas sourced from either a female or male fetus is believed to provideunique treatments for specific diseases such as the cases exhibited forerectile dysfunction and Peyronie's disease that would be benefitedgreatly by the use of female amniotic fluid based on the 49 growthfactors occurring in higher concentrations in that fluid source.Interestingly, females have a lower rate of Sudden Infant Death Syndrome(SIDS), and it is unknown why this is. Females also have a significantlyhigher amount of NP_001142.2 in their amniotic fluid that can preventSudden Infant Death Syndrome. One possible treatment of infants at highrisk for SIDS is female specific Amniotic Fluid Nebulizers and Inhalers.Alternatively, it is believed that male amniotic fluid would have betterresults treating or preventing other diseases, such as Pulmonary ChronicObstructive Pulmonary Diseases, or Wound healing. This is based upon thegene charting we did by putting these genes in certain functionalcategories for our abstract that was accepted to the Society of SexualMedicine Conference this year. In any event, it is clear that there aredistinctions in patient treatments based on the gender source of theamniotic fluid. As a result, the present invention is directed to thisability to source amniotic fluid based on gender of the source. Thisinvention can explain why sometimes physicians claim their amnioticfluid patient results are unreliable, when amniotic fluid is used as atreatment. It is likely dependent on the gender of the amniotic fluidfetus and the disease process being treated.

TABLE 1 Growth Factors, Proteins, Female Dominant Abundance Ratio P-Abundance Value: Ratio Adj. Growth Factors and (male)/ P-Value: Proteins(female) (male)/(female) NP_001306130.1 0.305 0.005510287 proteinS100-A8 isoform d [Homo sapiens] S100A8 NP_002956.1 0.278 0.002675392protein S100-A9 [Homo sapiens] S100A9 NP_065723.1 0.214 0.000273433protein S100-A14 [Homo sapiens] S100A14 NP_005611.1 0.258 0.001497062protein S100-A11 [Homo sapiens] S100A11 NP_005538.2 0.215 0.00029255involucrin [Homo sapiens] IVL XP_016870146.1 0.208 0.000211106 annexinA1 isoform X1 [Homo sapiens] ANXA1 NP_001531.1 0.3 0.004865982 heatshock protein beta-1 [Homo sapiens] HSPB1 NP_001303936.1 0.2320.000582268 protein S100-A16 [Homo sapiens] S100A16 NP_000415.2 0.2490.001099551 keratin, type II cytoskeletal 5 [Homo sapiens] KRT5NP_775109.2 0.214 0.000295615 keratin, type II cytoskeletal 6C [Homosapiens] KRT6C; KRT6A NP_057274.1 0.189 8.49595E−05 cornulin [Homosapiens] CRNN NP_002263.3 0.079 1.32454E−09 keratin, type IIcytoskeletal 4 [Homo sapiens] KRT4 NP_705694.2 0.075 6.08683E−10keratin, type I cytoskeletal 13 isoform a [Homo sapiens] KRT13NP_002267.2 0.16 1.40704E−05 keratin, type I cytoskeletal 19 [Homosapiens] KRT19 NP_005545.1 0.131 1.34666E-06 keratin, type IIcytoskeletal 6C [Homo sapiens] KRT6A NP_001435.1 0.254 0.001280949 fattyacid-binding protein, epidermal [Homo sapiens] FABP5 NP_000587.1 0.2480.001059966 insulin-like growth factor- binding protein 1 precursor[Homo sapiens] IGFBP1 NP_001276737.1 0.196 0.000120054 alpha-crystallinB chain isoform 1 [Homo sapiens] CRYAB NP_001313.1 0.206 0.000191209cystatin-SA precursor [Homo sapiens] CST2 NP_444513.1 0.232 0.000583814dermcidin isoform 1 preproprotein [Homo sapiens] DCD NP_001186757.10.212 0.000187342 cornifin-A [Homo sapiens] SPRR1A XP_016880078.1 0.2080.000211514 junction plakoglobin isoform X1 [Homo sapiens] JUPNP_002696.3 0.274 0.002423476 periplakin [Homo sapiens] PPLNP_001186652.1 0.269 0.002099494 cellular retinoic acid-binding protein2 [Homo sapiens] CRABP2 NP_004406.2 0.212 0.000257882 desmoplakinisoform I [Homo sapiens] DSP XP_016873759.1 0.292 0.003976564 neuroblastdifferentiation- associated protein AHNAK isoform X2 [Homo sapiens]AHNAK NP_002266.2 0.215 0.000291601 keratin, type I cytoskeletal 15[Homo sapiens] KRT15 NP_001005337.1 0.193 0.000103096 plakophilin-1isoform 1a [Homo sapiens] PKP1 XP_016881722.1 0.248 0.001052441choriogonadotropin subunit beta variant 1 isoform X1 [Homo sapiens] CGB1NP_056932.2 0.154 9.09917E−06 keratin, type II cytoskeletal 2 oral [Homosapiens] KRT76 NP_002855.2 0.269 0.002069415 pregnancy zone proteinprecursor [Homo sapiens] PZP NP_005969.1 0.163 1.22605E−05 proteinS100-A2 [Homo sapiens] S100A2 NP_000688.2 0.158 1.24344E−05 arachidonate12- lipoxygenase, 12S- type [Homo sapiens] ALOX12 NP_443112.2 1.2850.472931274 phosphoinositide-3- kinase-interacting protein 1 isoform 1precursor [Homo sapiens] PIK3IP1 NP_001979.2 0.275 0.002473144envoplakin isoform 2 [Homo sapiens] EVPL NP_001116437.1 0.1495.69435E−06 repetin [Homo sapiens] RPTN NP_057215.3 ras- 0.2990.004452985 related protein Rab-10 [Homo sapiens] RAB10 NP_001171712.10.225 0.000445982 voltage-dependent anion-selective channel protein 2isoform 1 [Homo sapiens] VDAC2 XP_005259194.1 0.294 0.003044342 40Sribosomal protein S16 isoform X1 [Homo sapiens] RPS16 NP_001142.2 0.2540.001107207 ADP/ATP translocase 1 [Homo sapiens] SLC25A4 NP_009140.1 60S0.178 3.5374E−05 ribosomal protein L35 [Homo sapiens] RPL35 NP_057671.20.229 0.00052652 chromobox protein homolog 3 [Homo sapiens] CBX3;C15orf57; CCDC32 NP_071404.2 p53 0.181 3.31804E−05 apoptosis effectorrelated to PMP-22 [Homo sapiens] PERP NP_001001973.1 0.277 0.002331947ATP synthase subunit gamma, mitochondrial isoform L (liver) precursor[Homo sapiens] ATP5C1 NP_114409.2 plasma 0.15 9.52609E−06alpha-L-fucosidase precursor [Homo sapiens] FUCA2 NP_001132929.1 B-0.272 0.001161581 cell receptor-associated protein 31 isoform a [Homosapiens] BCAP31 NP_001135757.1 40S 0.256 0.000823958 ribosomal proteinS24 isoform d [Homo sapiens] RPS24 NP_002943.2 40S 0.301 0.003440049ribosomal protein S2 [Homo sapiens] RPS2 NP_005637.3 0.271 0.00199776probable methyltransferase TARBP1 [Homo sapiens] TARBP1

With reference to table 1, this table shows and identifies 49 attributessuch as growth factors and proteins in a first column that are of ahigher proportion when gender specific amniotic fluid is from a femalefetal source. the adjacent column reflects a ratio of the male/femaleproportions each of the attributes. As shown, all are substantiallybelow 1. These listed growth factors and proteins are in a higherconcentration than from a male fetal source as shown in the chart. Theseare in the second column called: “Abundance Ratio P-Value:(male)/(female)” (AA). The third column called “Abundance Ratio AdjP-Value: (male)/(female)” (AB). In this column, each protein is measuredand adjusted to all 1451 proteins measured versus one individualprotein. It is compared to all other proteins such that there is norandom chance a specific one is elevated.

The P value used to determine statistically significant differences wasP<0.005. This is much more stringent than a usual P value of P<0.05.Some of these are over 10 times greater in female than male amnioticfluid.

TABLE 2 Abundance Ratio P- Abundance Value: Ratio Adj. Growth Factorsand (male)/ P-Value: Proteins (female) (male)/(female) NP_036246.1 4.370.000224368 caspase-14 precursor [Homo sapiens] CASP14 NP_002007.110.982 3.09098E−09 filaggrin [Homo sapiens] FLG NP_001153392.1 2.9660.002397936 vascular endothelial growth factor receptor 1 isoform 2precursor [Homo sapiens] FLT1 NP_001159921.1 2.769 0.0035595inter-alpha-trypsin inhibitor heavy chain H4 isoform 2 precursor [Homosapiens] ITIH4 NP_076956.1 2.979 0.005795033 gamma-glutamylcyclotransferase isoform 1 [Homo sapiens] GGCT NP_000377.1 4.0120.000497011 bleomycin hydrolase [Homo sapiens] BLMH NP_001289194.1 3.1240.002654632 neutrophil defensin 1 isoform 1 preproprotein [Homo sapiens]DEFA1B; DEFA1 NP_005971.1 protein 2.689 0.005140474 S100-P [Homosapiens] S100P NP_059118.2 5.621 1.70252E−05 calmodulin-like protein 5[Homo sapiens] CALML5 NP_001014364.1 6.251 5.14524E−06 filaggrin-2 [Homosapiens] FLG2 XP_005266835.2 3.945 0.000579305 serpin B12 isoform X1[Homo sapiens] SERPINB12 NP_001231367.1 3.528 0.001526005 arginase-1isoform 1 [Homo sapiens] ARG1 NP_009175.2 2.936 0.006442024 proline-richprotein 4 isoform 2 precursor [Homo sapiens] PRR4 NP_778253.2 keratin,4.349 0.000235064 type II cytoskeletal 1b [Homo sapiens] KRT77XP_011525801.1 26.879 1E−17 dermokine isoform X4 [Homo sapiens] DMKNNP_002768.3 4.328 0.000312229 myeloblastin precursor [Homo sapiens]PRTN3 XP_011525797.1 14.715 2.03593E−12 dermokine isoform X1 [Homosapiens] DMKN NP_775103.1 5.601 2.88672E−06 small proline-rich protein 4[Homo sapiens] SPRR4 NP_291031.2 3.142 0.001370691 guanylin precursor[Homo sapiens] GUCA2A NP_991403.1 3.435 0.001902331 lipolysis-stimulatedlipoprotein receptor isoform 2 [Homo sapiens] LSR NP_001138479.1 4.2170.000314174 mucin-7 precursor [Homo sapiens] MUC7 NP_002414.1 matrilysin3.949 0.000223214 preproprotein [Homo sapiens] MMP7 NP_002099.1 3.3740.002201512 histidine ammonia-lyase isoform 1 [Homo sapiens] HALXP_016864371.1 3.128 0.001518546 chloride intracellular channel protein6-like [Homo sapiens] LOC107986211 NP_000418.2 3.776 0.000853704loricrin [Homo sapiens] LOR NP_543145.1 13.268 2.77156E−12 WAPfour-disulfide core domain protein 12 precursor [Homo sapiens] WFDC12NP_001078851.1 2.985 0.005700476 proactivator polypeptide- like 1preproprotein [Homo sapiens] PSAPL1 XP_011546040.1 2.844 0.005070247major histocompatibility complex, class II, DR beta 1 isoform X1 [Homosapiens] HLA-DRB3; LOC100507709; LOC100507714; LOC105369230; HLA-DRB1NP_006323.2 gamma- 4.058 0.000140941 interferon-inducible lysosomalthiol reductase preproprotein [Homo sapiens] IFI30 NP_002621.1gastricsin 3.343 0.002374363 isoform 1 preproprotein [Homo sapiens] PGCNP_001963.1 neutrophil 4.989 6.04516E−05 elastase preproprotein [Homosapiens] ELANE XP_011534801.1 2.909 0.00365075 cathepsin G isoform X1[Homo sapiens] CTSG NP_001180262.1 coronin- 3.38 0.001023141 1A [Homosapiens] CORO1A NP_005441.1 noggin 4.593 4.59596E−05 precursor [Homosapiens] NOG NP_006240.4 basic 2.809 0.003799462 salivary proline-richprotein 3 precursor [Homo sapiens] PRB3 NP_001019850.1 skin- 2.8040.005355183 specific protein 32 [Homo sapiens] C1orf68 NP_001691.1azurocidin 4.412 7.21396E−05 preproprotein [Homo sapiens] AZU1XP_011541136.1 3.942 0.000115751 neutrophil collagenase isoform X1 [Homosapiens] MMP8 XP_005247417.1 3.005 0.00207313 lysosome-associatedmembrane glycoprotein 3 isoform X1 [Homo sapiens] LAMP3 NP_068741.1Fanconi 2.955 0.002748764 anemia group E protein [Homo sapiens] FANCENP_002143.1 sarcoplasmic 3.064 0.002018187 reticulum histidine-richcalcium-binding protein precursor [Homo sapiens] HRC NP_064581.2pre-mRNA- 3.954 0.000216534 splicing factor SYF1 [Homo sapiens] XAB2

As shown in table 2, is a second table showing and identifying the 42attributes such as growth factors and proteins that are in a higherproportion when gender specific amniotic fluid is from a male fetalsource than a female fetal specific source. In this case, the male fetalsource amniotic fluid has a higher percentage of these growth factorsand proteins when compared to a female fetal source amniotic fluid. Someof these are over 10 times greater in male than female amniotic fluid.As shown in table 2, the abundance ratio of male/female is substantiallygreater than 1.0.

With reference to FIG. 1, a table is shown for the PSV, wherein thefemale fetal source amniotic fluid was statistically significantlybetter than the male fetal source amniotic fluid when treating erectiledysfunction and improving Penile Peak Systolic Velocity as discussedabove.

It is believed that this discovery and resultant amniotic fluid productbased on gender can provide unique treatments. Such treatments can beprovided as ointment based for treating wounds and other soft tissue.Alternatively, treatments can be provided as a fluid for directinjection as was the case in the study on erectile dysfunction andPeyronie's' disease. The fluid can also be supplied as a nebulised oraerosolized treatment or suppository.

With reference to FIG. 2, an exemplary syringe 30 with needle 32 isshown with an amount of amniotic fluid 100 suitable injection based onthe gender of fetal source.

It is important to note that during the analysis of amniotic fluid thatthe growth factors exhibited are interesting, some dealing with suddeninfant death syndrome and may be useful in identifying precursors tosuch an event. Importantly, it is believed the ability to differentiategrowth factors of amniotic fluid based on gender of the fetal sourcewill be of value as it is applied to treating specific disease. Of note,most of the growth factors and cytokines deal with metabolic pathwaysand cell proliferation, cell differentiation, and cell death. As aresult, it is believed that a sterile supply of amniotic fluid based ongender may result in unique treatment protocols heretofore notattempted. The results revealed one growth factor predominantly more inFEMALES, XP_016881722.1, that is a Choriogonadotropin, which is believedto be important.

It is believed that a physician, knowing the differences in specificgrowth factors, can select from one or more of the gender differentiatedamniotic fluids to select the one with the highest concentrations ofthese more prevalent growth factors and proteins to achieve the desiredtreatment results. Heretofore this has not been done. This is similar tostem cell research where individual cells are noted by their expressedmarker. In this case the amniotic fluid is being distinguished based onits increased percentages of specific growth factors which would then beused to determine which gender based amniotic fluid can be used inparticular treatments. In other words, this unique discovery is thefirst step in determining targeted biological exosome and growth factortherapy for diseases. Since we are able to isolate in higherconcentrations specific growth factors and exosomes and cytokines withminimal manipulation of the Amniotic Fluid.

The composition and method of the present invention can be used incombination with compositions and methods in U.S. Ser. Nos. 15/158,101;14/363,142; and 15/541,988 all of which are being incorporated herein byreference in their entirety in the present invention.

With reference to FIGS. 3-9, graphs and volcano plots are shown thatillustrate differences between male and female samples of the genderspecific amniotic fluid, female is indicated by blue or F and male isindicated in red or M.

Variations in the present invention are possible in light of thedescription of it provided herein. While certain representativeembodiments and details have been shown for the purpose of illustratingthe subject invention, it will be apparent to those skilled in this artthat various changes and modifications can be made therein withoutdeparting from the scope of the subject invention. It is, therefore, tobe understood that changes can be made in the particular embodimentsdescribed which will be within the full intended scope of the inventionas defined by the following appended claims.

What is claimed is:
 1. A method of treating a patient with a tissuedisease or malformity comprises the steps of: identifying the tissueregion to be treated and selecting a location to apply either topicallyor by injection or inhalation a composition containing amniotic fluid;selecting the composition containing amniotic fluid wherein the amnioticfluid has a quantity of gender specific amniotic fluid based on a genderof a fetal source; and applying or injecting or inhaling the compositionat or into the selected location.
 2. The method of claim 1 wherein thegender specific amniotic fluid of a female fetal source and a male fetalsource exhibit a total of about 1500 growth factors and cytokines whentested under mass spectrometry.
 3. The method of claim 2 wherein themale fetal source amniotic fluid exhibits a number of growth factors ina higher concentration than the female fetal source amniotic fluid. 4.The method of claim 1 wherein each of the number of growth factorsconcentration higher in each gender specific amniotic fluid is greaterthan 25 and less than 50 of the total of about
 1500. 5. The method ofclaim 4 wherein the male fetal source amniotic fluid has 45 growthfactors statistically significantly greater than the female fetal sourceamniotic fluid.
 6. The method of claim 1 wherein the selection of thecomposition with amniotic fluid is guided by differences in growthfactors and cytokines between the gender specific amniotic fluids. 7.The method of claim 1 wherein the tissue disease or malformity is in apenis.
 8. The method of claim 1 wherein the tissue disease or malformityis wound healing and the gender specific amniotic fluid is a male fetalsource amniotic fluid.
 9. The method of claim 1 is pulmonary disease andthe gender specific amniotic fluid is gender specific amniotic fluid,either male fetal source amniotic fluid or female source amniotic fluid.10. A composition for treating a patient with a tissue disease ormalformity comprises a composition containing amniotic fluid wherein theamniotic fluid has a specific quantity of gender specific amniotic fluidbased on a gender of a fetal source which implies mixing and matchingdifferent amounts of the different amniotic Fluids based upon gender ofthe fetuses.
 11. The composition of claim 10 wherein the gender specificamniotic fluid of female fetal source and the male fetal source exhibitabout 1500 growth factors and cytokines when tested under massspectrometry.
 12. The composition of claim 11 wherein the male fetalsource amniotic fluid exhibits a number of growth factors in a higherconcentration than the female fetal source amniotic fluid.
 13. Thecomposition of claim 11 wherein each of the number of growth factorsconcentration higher in each gender specific amniotic fluid is greaterthan 25 and less than 50 of the total of about
 1500. 14. The compositionof claim 13 wherein the male fetal source amniotic fluid has 45 growthfactors statistically greater than the female fetal source amnioticfluid.
 15. The composition of claim 10 wherein the selection of thecomposition with amniotic fluid is guided by differences in growthfactors and cytokines and exosomes and Messenger MRNA between the genderspecific amniotic fluids.
 16. The composition of claim 15 wherein thegender specific amniotic fluid has a selected proportion of male fetalsource amniotic fluid or female fetal source amniotic fluid relative tothe total amniotic fluid.
 17. The composition of claim 16 wherein theselected proportion of male fetal source or female fetal source amnioticfluid is between 75 percent and up to 100 percent and at 100 percent theamniotic fluid is either all female fetal source or all male fetalsource amniotic fluid.
 18. The composition of claim 10 wherein theamniotic fluid is further selected based on disease process based uponage of term pre planned C-Sections in addition to gender of fetus. 19.The composition of claim 1 wherein 5 or more of the following growthfactors or proteins are present in the male fetal source amniotic fluid:NP_036246.1, NP_002007.1, NP_001153392.1, NP_001159921.1, NP_076956.1,NP_000377.1, NP_001289194.1, NP_005971.1, NP_059118.2, NP_001014364.1,XP_005266835.2, NP_001231367.1, NP_009175.2, NP_778253.2,XP_011525801.1, NP_002768.3, XP_011525797.1, NP_775103.1, NP_291031.2,NP_991403.1, NP_001138479.1, NP_002414.1, NP_002099.1, XP_016864371.1,NP_000418.2, NP_543145.1, NP_001078851.1, XP_011546040.1, NP_006323.2,NP_002621.1, NP_001963.1, XP_011534801.1, NP_001180262.1, NP_005441.1,NP_006240.4, NP_001019850.1, NP_001691.1, XP_011541136.1,XP_005247417.1, NP_068741.1, NP_002143.1, NP_064581.2.
 20. The Method ofclaim 6 wherein the disease is Sudden Infant Death Syndrome or any otherPulmonary Disease.